Cancer kills one American in four. Genetic instability is a hallmark of cancer;it results from mutations that increase the rate of genetic change and accelerates cancer progression and the generation of mutant cells that resist chemo- and radiation-therapy. Genetic instability is manifested through elevated rates of point mutation, chromosome loss and gain, and chromosomal rearrangements. The inaccessibility of the early stages of cancer evolution makes it hard to determine when genetic instability arises and how it contributes to tumor progression. The goal of this project is to model these processes by evolving the budding yeast, Saccharomyces cerevisiae, to inactivate multiple growth suppressor genes: genes whose active form makes cells sensitive to growth suppressing drugs. In diploid yeast, growth suppressor genes mimic tumor suppressor genes because both copies of the gene must be inactivated to cause increased cell proliferation. Genetic analysis, comparative genomic hybridization, and genome sequencing will be used to follow the evolution of genetic instability, find the mutations that cause it, and understand its mechanism. Genetic instability will be studied and in haploid and diploid yeast evolved to inactivate four different growth suppressor genes under two types of selection: sequential exposure to high concentrations of each growth suppressing drug (strong, sequential selection) and simultaneous exposure to low concentrations of all four growth suppressing drugs (weak, simultaneous selection). The experiments are designed to address four specific aims. 1) Does strong, sequential selection increase the point mutation frequency in haploid strains? 2) What forms of genetic instability results from strong, sequential selection for loss of heterozygosity in diploid cells? 3) How does this differ from the outcome of strong, sequential selection for loss of function in both copies of growth suppressor genes? 4) How different are the outcomes of simultaneous, weak selection and sequential, strong selection? Cancer is an evolutionary disease: genetic changes allow cells to grow and divide inappropriately and evade anti-cancer treatments. All cancers are genetically unstable, meaning that they have acquired mutations that make other mutations more likely. This project will use brewer's yeast to study how cells evolve to become genetically unstable, with the aim of improving cancer diagnosis and therapy.